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Abstract:

The present invention cloned melanin biosynthesis genes encoding
polyketide synthase (PKS), scytalone dehydratase (SCD) and
1.3.8-trihydroxynaphthalene reductase (THN) from the dematiaceous
Alternasia alternate into plasmid pCAMBIA1300, followed by transformation
of the plasmid into Matarhizium anisopliae via Agrobacterium
tumefaciens-mediated transformation. The transformant was able to express
the abovementioned genes and synthesize melanin, which then showed
enhanced UV tolerance. The transcription and expression of these melanin
genes were confirmed in several pathways. The tolerances toward UV
radiation, drought and high temperature were increased significantly in
these transformants. In addition, the host insects were more susceptible
to these transformants under UV radiation.

2. The fungi transformant as claimed in claim 1, wherein the PKS gene in
the genomic DNA of the fungi transformant is inserted through a
transforming mediator having a PKS gene vector.

3. The fungi transformant as claimed in claim 2, wherein the transforming
mediator is Agrobacterium tumefaciens.

4. The fungi transformant as claimed in claim 2, wherein the PKS gene
vector is plasmid pCAMBIA PKS-ORF, which comprises at least one PKS gene
and a promoter, and the promoter operably linked to the PKS gene.

5. The fungi transformant as claimed in claim 4, wherein the PKS gene is
subcloned from Alternaria alternata.

6. The fungi transformant as claimed in claim 1, wherein the SCD gene in
the genomic DNA of the fungi transformant is inserted through a
transforming mediator having a SCD gene vector.

7. The fungi transformant as claimed in claim 6, wherein the transforming
mediator is Agrobacterium tumefaciens.

8. The fungi transformant as claimed in claim 6, wherein the SCD gene
vector is plasmid pCAMBIA Scy, which comprises at least one SCD gene and
a promoter, and the promoter operably linked to the SCD gene.

9. The fungi transformant as claimed in claim 8, wherein the SCD gene is
subcloned from Alternaria alternata.

10. The fungi transformant as claimed in claim 1, wherein the THN gene in
the genomic DNA of the fungi transformant is inserted through a
transforming mediator having a THN gene vector.

11. The fungi transformant as claimed in claim 10, wherein the
transforming mediator is Agrobacterium tumefaciens.

12. The fungi transformant as claimed in claim 10, wherein the THN gene
vector is plasmid pCAMBIA THN, which comprises at least one THN gene and
a promoter, and the promoter operably linked to the THN gene.

13. The fungi transformant as claimed in claim 12, wherein the THN gene
is subcloned from Alternaria alternate.

14. A method for using a fungi transformant as a biocontrol agent,
wherein the fungi transformant as claimed in claim 1 was applied to
express melanin biosynthesis proteins of PKS, SCD and THN to increase the
infection of the fungi transformant in a host under a stress condition.

15. The method as claimed in claim 14, wherein the stress condition is a
UV-radiation environment.

16. The method as claimed in claim 14, wherein the stress condition is a
drought environment.

17. The method as claimed in claim 14, wherein the stress condition is a
high temperature environment.

18. The method as claimed in claim 14, wherein the host is an insect.

19. A vector expressing melanin biosynthesis proteins, wherein the vector
comprises at least one melanin biosynthesis gene and a promoter, and the
promoter operably linked to the melanin biosynthesis gene.

20. The vector as claimed in claim 19, wherein the vector is plasmid
pCAMBIA PKS-ORF (BCRC 940577), and the melanin biosynthesis gene is a
polyketide synthase (PKS) gene.

21. The vector as claimed in claim 19, wherein the vector is plasmid
pCAMBIA Scy (BCRC 940578), and the melanin biosynthesis gene is a
scytalone dehydrtase (SCD) gene.

22. The vector as claimed in claim 19, wherein the vector is plasmid
pCAMBIA THN (BCRC 940579), and the melanin biosynthesis gene is a
1,3,8-trihydroxynaphthalene reductase (THN) gene.

Description:

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a microorganism biocontrol agent,
in particular to a fungi transformant containing melanin biosynthesis
genes to increase the tolerance to environmental stress and its method.

[0003] 2. The Prior Arts

[0004] Beneficial microorganisms such as Metarhizium spp., Beauveria spp.,
Trichoderma spp. and Bacillus spp. have been applied in pest management
for a long time. There are numerous studies showing positive effects in
pest control with beneficial microorganisms in field application so far
(Genthner et al., 1998). However, the stability and reproducibility of
the studies were limited in field application due to the factors of
stressful environmental condition. (Alves et al., 1998; Hedgecock et al.,
1995). Natural environmental factors such as high temperature, drought
and UV radiation are the bottle necks for field application (Fargues,
1996). Therefore, it becomes an important issue to protect the beneficial
microorganisms to lower the stress of the environment.

[0005] The survival of biocontrol microorganisms in field during the
contact and application period for the strain of biological control
microorganism and the targets (such as pest or pathogens) is important
for tolerance of stressful environment. The application effects won't be
stable for those that can not overcome the barriers and survive in field.
Therefore, the improvement of tolerance to stressful environment for the
biocontrol microorganism is an important issue. At present, the
improvement is employed through formulation. Hedimbi et al (2008) used
olive oil containing commercial sunscreens as additive to treat
Metarhizium anisopliae conidia, and exposed them to an artificial UV
source for up to 5 hours. Survival of conidia in oil formulation was
around 29-40% while in control (water) was 4%. The conidial germination
rates produced from mycelium were lowered to 20% when irradiated with
UV-A for 6 hours (Rangel et al. 2008). The increased osmotic stress also
made the germination rate of M. anisopliae dropped sharply. Bacillus
thuringiensis is a biocontrol agent other than eukaryotes. The
δ-endotoxin produced in B. thuringiensis is easily degraded by
UV-radiation. The activity of B. thuringiensis is lost under short term
exposure of sunshine. Previous studies have shown that the UV tolerance
was increased in melanin producing B. thuringiensis strains. To overcome
the stressful conditions mentioned above, the adjustment on application
time (to avoid the strong light), formulation change (such as using oil
to overcome the drought problem) or adding sunscreen to protect the
microorganisms were employed. In addition, nutritional stress has been
applied to increase the tolerance to high temperature, low water content
and UV radiation. Trehalose and mannitol levels were accumulated in M.
anisopliae conidia after nutritional stress, which may be the reason for
high tolerance to the stressful condition (Rangel et al., 2008).

[0006] Biosynthesis of DHN melanin is synthesized by a polyketide pathway,
through the genes encoding Polyketide synthase (PKS), Scytalone
dehydrtase (SCD), and 1,3,8-trihydroxynaphthalene reductase (THN). It
started with a PKS using malonyl-CoA as a substrate to produce
1,3,6,8-tetrahydroxynaphthalene, 1,3,6,8-THN (Fujii et al., 2000),
followed by reducatse catalysis to produce scytalone, dehydration by SCD
to yield 1,3,8-trihydroxynaphthalene (1,3,8-THN), reduction by THN to
yield vermelone, dehydration to produce melanin precursor
1,8-dihydroxynaphthalene (1,8-DHN), and then oxidation and polymerization
to yield melanin.

[0007] Melanin is polymer existed broadly in organisms in nature and has a
variety of biological functions (Hill et al., 1992). It is negatively
charged, hydrophobic pigment with high molecular weight, which is formed
by the oxidative polymerization of phenolic and/or indolic compounds. In
many organisms, melanin protects cells from stressful conditions, such as
oxidation, extreme temperature, UV radiation, chemical, and biochemical
stresses (reviewed in Nosanchuk and Casadevall, 2003; Bell and Wheeler,
1986). Therefore, the use of melanin in the invention is a solution to
protect biocontrol agents.

SUMMARY OF THE INVENTION

[0008] Microorganisms such as M. anisopliae used as the biocontrol agents
need to tolerate the environmental stresses during the period to interact
with target organisms (pests or pathogens). The survival of the
biocontrol agent is quite important in the field. The activity would be
unstable if the abovementioned barriers were not overcome. It remains to
be an important issue to improve the stress tolerance of the biocontrol
agents.

[0009] The objective of the present invention is to provide a vector
expressing melanin biosynthesis proteins, a transforming mediator having
the vector, and a fungi transformant expressing melanin biosynthesis.

[0010] The fungi transformant of the present invention is M. anisopliae
transformant which was deposited in the Culture Collection and Research
Center (CCRC) of Taiwan with an accession number BCRC940577 on Dec. 24,
2009. The vectors were plasmids pCAMBIA PKS-ORF, pCAMBIA Scy and pCAMBIA
THN which were also deposited under the accession number BCRC 940577,
BCRC 940578 and BCRC 940579 respectively.

[0011] Another objective of the present invention is to provide a method
for preparing a fungi transformant expressing melanin biosynthesis, which
may be made through the following example, but is not limited to the
materials and steps mentioned below.

[0012] Yet another objective is to provide applications of biocontrol
agents using fungi transformants.

[0013] A technique has been employed to solve the problems of the prior
art, where the melanin biosynthesis genes from Alternaria alternate has
been cloned and transferred into the beneficial microorganisms to protect
the target microorganisms. The beneficial microorganism in the present
invention is M. anisopliae which could parasitize in many major pests.
The melanin biosynthesis genes from Alternaria alternate include genes
encoding polyketide synthase (PKS), scytalone dehydrtase (SCD), and
1,3,8-trihydroxynaphthalene reductase (THN). M. anisopliae is able to
synthesize melanin after these three genes were cloned into the A.
tumefaciens plasmid pCAMBIA 1300 respectively, followed by A.
tumefaciens--mediated transformation into M. anisopliae. The melanin can
be produced in a non-melanin producing fungi through the cloning and
transformation of DHN melanin biosynthesis gene.

[0014] The transformants of M. anisopliae according to the present
invention can effectively increase the tolerance to UV radiation, extreme
temperature or low temperature, and drought. The protective method using
microorganisms to synthesize melanin directly, which is different from
the traditional way by adding anti-UV compounds to microorganisms,
increases the survival potential, shows higher and faster infection
ability in stressful environment, further increases the application
efficiency of beneficial microorganisms. In the future, the present
invention can be applied in other beneficial microorganisms and plants to
enhance the tolerance of stressful conditions.

[0015] The present invention is further explained in the following
embodiment illustration and examples. Those examples below should not,
however, be considered to limit the scope of the invention, it is
contemplated that modifications will readily occur to those skilled in
the art, which modifications will be within the spirit of the invention
and the scope of the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The related drawings in connection with the detailed description of
the present invention to be made later are described briefly as follows,
in which:

[0017] FIG. 1 is the flowchart for cloning of DHN melanin biosynthesis
gene from A. alternate;

[0035] The term "biocontrol microorganisms or biocontrol agent" as used
herein refers to microorganisms or the active components used for pest or
pathogen control, which are formulated as a product and the sources of
microorganism include bacteria, fungi, virus and protozoa.

[0036] The term "stressful environment" as used herein refers to
unfavorable conditions such as UV-radiation, drought, extreme high or low
temperature to the survival of microorganism.

[0037] The term "transformant" as used herein refers to an organism that
has undergone transformation to receive foreign genes.

[0038] The term "vector" as used herein refers to a nucleic acid molecule
capable of self-duplicating and receiving foreign genes by insertion, and
transporting foreign genes into a receiver DNA through transformation.

Example 1

(1) Cloning of Melanin Biosynthesis Genes

[0039] Referring to FIG. 1, the flowchart for cloning of DHN melanin
biosynthesis gene from A. alternata BCRC30501 was shown. The detailed
steps for cloning of the genes encoding Polyketide synthase (PKS),
Scytalone dehydrtase (SCD), and 1,3,8-trihydroxynaphthalene reductase
(THN) were described below.

(2) Strains and Vectors

[0040] The fungi including A. alternata BCRC30501 and M. anisopliae
BCRC35505 used in the present invention were obtained from Food Industry
Research and Development Institute, Taiwan. Binary vector pCMABIA 1300
(NCBI gi:7638064) (Canberra, Australia) was used as backbone for plasmid
construction (Hajdukiewicz et al., 1994).

(3) Establishment of Genomic DNA Library

[0041] The mycelium of A. alternate was harvested after cultivation for
DNA extraction (according to Al-Samarrai, 2000). The genomic DNA library
of A. alternate was obtained with the kit of CopyControl® Fosmid
library production kit (Epicentre, Madison, Wis., USA). The clones were
dotted on a nylon filter for screening. 7000 colonies were dotted in
duplicate on nylon filter (22×22 cm) using a robotic arm for
Southern blot analysis.

[0043] The results of PCR amplification in electrophoresis are shown in
FIG. 2. FIG. 2A displayed a 700 bp fragment after PCR using primer pair
of KS1 and KS2. FIG. 2B displayed a 250 bp fragment after PCR using
primer pair of scyA and scyB. FIG. 2C displayed a 750 bp fragment after
PCR using primer pair of 1,3,8-tri(A) and 1,3,8-tri(B).

[0044] The PCR products were cloned into pGEM®-T (Promega, Wis., USA)
and analyzed the sequences. The melanin biosynthesis gene sequences were
confirmed with the NCBI GenBank with BLAST. DIG labeling probe was
produced with the abovementioned primer pairs and PCR DIG probe synthesis
kit (Roche, USA). The following ingredients were added respectively and
mixed thoroughly before PCR amplification. The plasmids in the reaction
were obtained from PCR product and pGEM-T cloning.

[0045] The DIG probes were used as probes for Southern Blot analysis with
the Fosmid library of A. alternate. The clone aaf01018E was found to
contain PKS and THN encoding genes, which was used in Shotgun library
construction. The Fosmid clone was analyzed for the complete sequence.

(6) Shotgun Library Construction

[0046] The genomic DNA of clone aaf01018E was sheared into small fragments
with Hydroshear (Gene Machine), and the ends were trimmed with Bal 31
nuclease and T4 DNA polymerase. The 2 kb-3 kb fragments of the
abovementioned DNAs were separated after electrophoresis and recovered
with QIAquick Gel Extraction kit (Qiagen). The recovered DNAs were
subcloned into pUC18 vector and transformed into E. coli DH5α (Life
Technologies) competent cells through heat shock with ampicillin
selection and X-gal screening. The DNA of the transformant was purified
and sequence analyzed by Big dye terminator ver 3.1 (Applied Biosystems)
using ABI 3730x1 DNA analyzer. The sequencing primers used were:

[0047] The sequences obtained were assembled with a Phred/Phrap/Consed
software developed in University of Washington (Phrap version 0.990329,
and Consed version 13) to yield a 40 kb sequence.

(7) Reverse Transcription PCR(RT-PCR)

[0048] Total RNA of A. alternate was extracted and treated with DNase. To
one μl of the treated RNA, 0.3 μl of 10 μM of dT18 mer, 1.0
μl of 10 mM dNTP and 10.7 of DEPC treated ddH2O were added and
mixed at 65° C. for 5 min. Then 4 μl of 5× First-strand
buffer, 1 μl of 0.1 M DTT (1,4-dithiothreitol), 1 μl of RNase out
and of SuperScript III reverse transcriptase (Invitrogen) were added and
mixed at 50° C. for 35 min followed by 70° C. for 15 min to
stop the reverse transcriptase reaction. PCR amplification was carried
out with this cDNA as template using ScyA, ScyB and 1,3,8-tri(A),
1,3,8-tri(B) primer pair to generate cDNA fragments of scytalone
dehydratase (SCD) and 1,3,8-trihydroxynaphthalene reductase (THN). These
cDNA fragments can be used for primer design in rapid amplification of
cDNA (RACE) after these sequences were determined.

(8) Rapid Amplification of cDNA Ends (RACE)

[0049] The RACE of SCD and THN genes were carried out with GeneRacer®
kit (Invitrogen, USA) according to the manual.

[0050] The cDNA obtained from GeneRacer® kit was used again for RACE.
The primers were designed after the abovementioned SCD and THN gene
sequences were determined:

[0052] Genes encoding PKS and THN were obtained during Fosmid cloning. The
full length of SCD gene was not cloned from Fosmid clone screening.
Therefore the cDNA of SCD obtained from RACE was used for primer pair
designation, probe preparation and Southern blot analysis. The DNAs of
clones containing SCD gene were purified and sequence determined with
primer walking method to determine the full length SCD genomic DNA
sequence. The primer set used were Scy_N--2-1: 5'
gCTACgAATgggCAgACAg 3' (SEQ ID NO: 13) and Scy_N--2-2: 5'
CCTCggCgAAgACCTTg 3' (SEQ ID NO: 14).

[0053] The full length gene sequences of SCD and THN were determined after
cDNA cloning and genome sequence analysis (FIGS. 4A and 4B). The SCD gene
has 775 bases with 2 introns of 48 bases and 49 bases respectively; while
the THN gene has 824 bases with 2 introns of 51 bases and 49 bases
respectively.

(10) Full Length DNA Sequence of PKS

[0054] cDNA clone of PKS was not obtained from previous experiment.
Therefore we used the genomic DNA for PKS cloning. The PKS containing
Fosmid yield DNA of 40 kb in length. This sequence was compared to the
GenBank data base using Basic Local Alignment Search Tool (BLAST) to
determine the range of the full length PKS. The region was then compared
with Propom search utility to define the functional motifs (FIG. 3) and
redefine the range. The functional motifs were shown in FIG. 3 (KS:
β-keto synthase motif; AT: acyl transferase motif; ACP: acyl carrier
protein motif and TE: thioesterase).

[0055] Restriction enzyme AvrII showed a single site in the PKS gene range
after analysis. The primer pair was designed according to this single
site. PCR amplification was performed with the template of PKS containing
Fosmid, and a primer pair to yield a 3-kb fragment which was designated
PKS-Sbf. The primer set used were:

[0057] From the example of the present invention, the gene fragment of SCD
obtained was 775 bases with 2 introns of 48 bases and 49 bases
respectively; and the gene fragment of THN gene was 1104 bases with 2
introns of 51 bases and 49 bases respectively. The PKS gene fragments
include PKS-Asc (3.5 kb) and PKS-Sbf (3 kb)

Example 2

(1) Construction of Transformation Vectors

[0058] Referring to FIG. 5, the flowchart for establishing transformant of
M. anisopliae and the analysis of physiology and biochemistry. First, the
binary vector was constructed according to the present invention. Then
these 3 genes were inserted into binary vector pCAMBIA 1300 respectively
for Agrobacterium-mediated transformation.

(1) Construction of THN Gene Harboring Binary Vector

[0059] Plasmid pCAMBIA was used as binary vector backbone for
transformation. The selection marker is HygromycinR and reporter
gene is green fluorescent protein (GFP), under the control of
glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter from Aspergillus
nidulans. PCR amplification was performed with the template of pAN7-1
(NCBI gi: 475166), primer set of BstXI-GPD-s and Hyg-XhoI-a to clone the
GPD promoter and HygromycinR gene. The primer set was designed to have a
BstXI site in GDP promoter region and an XhoI site in Hygromycinr gene.
The PCR product generated is 1.9 kb long and subcloned into pGEM-T easy
to yield pGEM-GH.

[0060] The 35S promoter and CDS3 (Hygromycin resistant gene) were removed
by BstXI and XhoI restriction enzyme digestion from plasmids pCAMBIA1300
and pGEM-GH, followed by ligation of 1.9 kb of GPD-HygromycinR from
pAN7-1 and resulted in pCAM-GH as the left border of the binary vector.

(b) Construction of Right Border of the Binary Vector

[0061] Two primer set was designed to contain a GDP promoter and a TrpC
terminator, restriction sites in the middle and both ends. PCR
amplification was performed with the template of pAN7-1 (NCBI gi:
475166), and the above-mentioned primer sets to ligate these 2 fragments.
The sequences of the primers were:

[0062] Primer set of GPD-K-s and GPD-S-A-a were used to amplify amplicon
of GPD promoter; while primer set of Trp-S-A-s and Trp-H-a were used to
amplify the TrpC terminator. Both PCR products were mixed as primers for
each other, followed by addition of dNTP, PCR buffer and Taq DNA
polymerase for 5 cycles. Final amplification was carried with primers
GPD-K-s and Trp-H-a (refers to FIG. 6A). The PCR product was cloned into
pGEM-T easy to yield pGEM-GT.

[0063] The plasmids pGEM-GT and pCAM-GH were digested with KpnI and
HindIII. Then the fragment containing GDP promoter and TrpC terminator
was ligated to pCAM-GH to yield pCAM-GH-GT.

[0064] Primer set was designed to have a full length of THN cDNA and
restriction sites of SbfI and AscI. The sequence of primers are: Tri-S-s
(SEQ ID NO: 25): 5'-CTGAAGGCCTGCAGGTCATCACAACCACTCTCATCAC-3' and Tri-A-a
(SEQ ID NO: 26): 5'-TTATTGGCGCGCCGTGCTTAAACGTTTCATTATCT-3'. The PCR
product was cloned into pGEM-T easy to yield GEM-TriFL.

[0065] Plasmids pGEM-TriFL and pCAM-GH-GT were digested with SbfI and
AscI. The full length cDNA of THN was ligated into pCAM-GH-GT to yield
pCAM-GH-GT-Tri. This complete binary vector, which contains THN gene and
can be used for transformation (FIG. 8C), was deposited in Culture
Collection and Research Center (CCRC) of Taiwan with an accession number
BCRC 940579.

(2) Construction of SCD Gene Harboring Binary Vector

(a) Construction of Left Border of the Binary Vector

[0066] The GPF (green fluorescent protein) gene were PCR amplified with
pRF280 (Toews et al., 2004) as template to yield a 700 bp fragment. Part
of the 3'-end sequence of GDP was added into the 5'-end of the sense
primer; and an XhoI site was added in the 3'-end of the antisense primer.
The sequence of primers are: GDP-GFP-s (SEQ ID NO: 27):
5'-ACATCACCATGGTGAGCAAGGG CGAGGAGCTGTTCAC-3' and GPD-GFP-XhoI-a (SEQ ID
NO: 28): 5'-ATAGGCCTCGAGTCTATTTGTACAGCTCGTCCATGCC-3'. The DGP promoter
was amplified with the following primer set to yield a 1000 bp fragment.
BstXI-GPD-s: 5'-ATGACCAGCATGTTGGCTCCGCCGCCTCCACCATTTGTA-3' (the same as
SEQ ID NO: 19) and GDP-GFP-a: 5'-CTTGCTCACCATGGTGATG
TCTGCTCAAGCGGGGTAGCT-3' (SEQ ID NO: 29).

[0067] Both PCR products (700 bp for GFP and 1000 bp for GPD) were mixed
as primers for each other, followed by addition of dNTP, PCR buffer and
Taq DNA polymerase for 5 cycles. Final amplification was carried with
primers BstXI-GPD-s and GPD-GFP-XhoI-a (refers to FIG. 6B). The 1.7 kb
PCR product was cloned into pGEM-T easy to yield pGEM-GF.

[0068] Plasmids pGEM-TGF and pCAMBIA-1300 were digested with BstXI and
XhoI. The fragment containing GPD promoter and GFP partial sequence was
ligated with pCAMBIA to yield pCAM-GF.

(b) Construction of Right Border of the Binary Vector

[0069] Plasmids pGEM-GT and pCAM-GF were digested with KpnI and HindIII.
The fragment containing GPD promoter and TrpC terminator partial sequence
was ligated with pCAM-GF to yield pCAM-GF-GT. A primer set was designed
to contain the full sequence of SCD cDNA and SbfI and AscI restriction
site in the end. The PCR product was cloned into pGEMT-easy to yield
pGEM-ScyFL. The sequences of the primer set are: Scy-S-s (SEQ ID NO: 30):
5'-CTGAAGGCCTGCAGGCAGTTTAAACATCTCCCACGA-3' and Scy-A-a (SEQ ID NO: 31):
5'-TTATTGGCGCGCCGGTCAAGCCTATCATTGTTCGTA-3'.

[0070] Plasmids pGEM-ScyFL and pCAM-GF-GT were digested with SbfI and
AscI. The full length cDNA of Scy was ligated into pCAM-GF-GT to yield
pCAM-GH-GT-Scy. This complete binary vector (FIG. 8B), which contains SCD
gene and can be used for transformation, was deposited in Culture
Collection and Research Center (CCRC) of Taiwan with an accession number
BCRC 940578.

(3) Construction of Pks Gene Harboring Binary Vector

[0071] Referring to FIGS. 7A-7E, the flow chart for constructing PKS gene
transforming vector. The PKS-Asc fragment was cloned into pGEM-T easy to
yield pPKS-Asc. This plasmid was digested with AscI and SbfI then ligated
with PKS-Sbf to yield pPKS-ORF, which contains the open reading frame
(ORF) of PKS.

[0072] Plasmid pCAM-GF-GT-Scy was digested with SbfI and AscI to remove
SCD cDNA. pPKS-ORF was also digested with SbfI and AscI to remove the PKS
full length gDNA. The full length gDNA of PKS was ligated into
pCAM-GF-GT-Scy to yield pCAM-PKS-ORF. This complete binary vector (FIG.
8A) was deposited in Culture Collection and Research Center (CCRC) of
Taiwan with an accession number BCRC 940577.

[0074] The co-cultivation medium was the same as IM, except the glucose
concentration of 5 mM was used, and the plate was prepared after the
addition of 1.5% agar.

(2) Preparation of Agrobacterium Tumefaciens

[0075] Agrobacterium tumefaciens EHA105 containing pCAMIBATri, pCAMBIA Scy
and pCAMBIA PKS-ORF (electroporation or tri-parental mating method can be
used) was cultivated in 10 ml of LB broth containing 50 μg/ml
kanamycin at 28° C., 220 rpm for 18 h. The cells were washed with
IM for three times after the centrifugation of 8,000 rpm for 5 min, and
resuspended into IM till O.D.600=0.30. 10 ml of the culture in IM
containing 50 μg/ml kanamycin and acetosyringone was cultivated at
28° C., 220 rpm till O.D.600=0.6-0.8.

[0076] M. anisoplia was cultivated in PDB at 25° C. in the dark for
2 days. The mycelia were removed with Miracloth®. The conidia were
collected after centrifugation at 5,000 rpm for 5 min, aspiration of the
medium and resuspension in sterile water at the concentration of 106
conidia/ml.

(3) Tranformation

(a) Co-Cultivation

[0077] The conidial suspension of M. anisopliae was mixed with
Agrobacterium tumefaciens containing PKS, SCD and THN respectively. And
100

[0078] 1 of the mixture was spread to the semi-permeable membrane covered
co-cultivation solid medium for cultivation at 28° C. for 2 days.
The semi-permeable membrane was cut into 1 cm width with a sterile knife
and transferred into CPZ medium (Difco) containing 250 μg ml-1
cefotaxime and hygromycein (100 μg/ml), with the conidium-containing
side facing down and 1 cm apart. The colonies obtained after 7 days in
the surface of the membrane were transferred again into CPZ medium
(Difco) containing 250 μg ml-1 cefotaxime and hygromycein (100
μg/ml).

(c) Screening of Transformant

[0079] The M. anisopliae transformant MA05-169 was deposited in Culture
Collection and Research Center (CCRC) of Taiwan on Dec. 24, 2009, with an
accession number BCRC 930124. The screening steps were described below.

[0080] The candidate strains in CPZ medium were subcultured first, then
the residual mycelia was collected in a 2 ml tube, followed by addition
of steel beads and shaking at 1300 rpm to break down the cells. The
genomic DNA was extracted with Maxwell® 16 genomic DNA Purification
Kits (Promaga, USA). PCR amplification was performed with this genomic
DNA and primers of PKS-TE-sen, PKS-TE-anti, Scy_N--2-1,
Scy_N--2-2 and Tri (A), Tri (B) to confirm the melanin synthesizing
gene in the candidate strains. Transformants containing melanin
synthesizing gene were cultivated in PDA medium containing hygromycein
(100 μml-1). PCR amplification was carried out again after 5
times of sub-culture to confirm the existence of each gene. The M.
anisopliae transformant MA05-169 was selected for the following
experiment. The colony morphology of wild type M. anisopliae MA35505 and
transformant MA05-169 were shown in FIG. 9, with the former on the left
and the latter on the right.

(d) Southern Blot Hybridization

[0081] The genomic DNAs of A. alternate, M. anisopliae MA35505 and M.
anisopliae MA05-169 were prepared according to Al-Samarrai et al (2000).
Restriction enzyme digestion with Hind III was carried out with SCD and
THN detection, and PstI was carried out with PKS detection. The digested
DNA was separated by electrophoresis in a 0.8% agarose at 50 v for 6 h,
and transferred to nylon membranes according to the procedures in
Molecular cloning (Sambrook and Russell, 2001). Hybridization probes were
synthesized with PCR amplification and labeled with DIG according to the
manufacturer's instruction (Roche). The sequences of primers were:

[0083] Total RNA of A. alternate, M. anisopliae MA35505, and M. anisopliae
MA05-169 was extracted according to the instruction of Trizol®
(Molecular Research Center, Inc). The extracted RNA was treated with 0.1
fold of 10×TURBO DNase buffer and 1 μl of TURBO DNase (Ambion)
at 37° C. for 30 min, followed by the addition of 0.1 fold of
TURBO inactivation reagent at room temperature for 2 min. The supernatant
was collected in a 1.5 ml of tube after the centrifugation of 10,000 rpm
for 1.5 min. 3 μl of the RNA was transfer to a quartz cuvette to
determine the concentration.

[0085] RT-PCR products were purified with Wizard® SV Gel and PCR
clean-up System (Promega) and stored at -20° C.

(4) PCR Amplification

[0086] The cDNA was using as template to perform PCR with primer pairs of
PKS-TE-sen, PKS-TE-anti, Scy_N--2-1, Scy_N--2-2,1,3,8-tri-(A)
and 1,3,8-tri-(B). GPD--456 primer set was designed according to the
sequence of M anisopliae glyceraldehyde-3-phosphate dehydrogenase mRNA
(NCBI gi|115607610) and using cDNA of M. anisopliae MA35505 and MA05-169
as templates for PCR amplification. In addition, GPD AA primer set was
also designed according to the sequence of A. alternata glyceraldehyde
3-phosphate dehydrogenase (NCBI gi|131747098) and using cDNA of A.
alternata as template for PCR amplification. The sequences of the primers
were:

[0088] A. alternate, M. anisopliae MA35505, and M. anisopliae MA05-169
were cultivated in PDB at 28° C. with shaking at 220 rpm. 100 ml
of the cell culture was incubated in 250 ml flasks. The mycelia were
collected after filtration. The melanin was extracted and purified with
strong base and strong acid according to Goncalves et al. (2005).

[0090] The electron paramagnetic resonance spectrophotometry (EPRS)
technique can also called as Electron Spin Resonance (ESR), which can be
used to evaluate quantitatively and qualitatively the presence of free
radicals. EPRS can be used to detect melanin based on the presence of
endogenous stable free radicals in melanin pigments.

[0091] Solid melanin was studied in the experiment. Each sample was
analyzed on 10 mg. EPR spectra were obtained with a Bruker EMX 10/12
spectrometer operating at modulation of 77° K, 9.48 GHz and 100
kHz (Enochs et al., 1993). The spectra were processed using Bruker
WIN-EPR® software version 2.11 (Bruker, Germany) to determine the
g-value (as shown in FIG. 12). g-Values of 2.00337 were found for both
samples, which indicated a free electron (Motoji, 1993). Therefore
melanin is postulated to have unpaired electrons.

(7) UV-Vis Spectrophotometry

[0092] The absorbance of purified melanin was determined by dissolving
melanin in 0.1 m boric buffer (pH 8.0) to the concentration of 0.002%
(w/v) and scanned in a UV-Vis spectrophotometer at wave length in the
range of 200-500 nm. 0.1 M of boric buffer was used as blank control
(Meredith and Riesz, 2004; Selvakumar et al., 2008).

[0093] The spectra plot was shown in FIG. 13. UV-Vis absorbance spectra
for standard DOPA-melanin and the 2 samples exhibited a similar pattern
with a typical peak at 230 nm, followed by decreasing linearly to the
basic absorbance at 500 nm. FIG. 14 showed the curves and slopes after
applying linear regression to log-spectra. The slope of regression line
for DOPA-melanin was -0.00209, and the slopes for the 2 samples from M.
anisopliae MA05-169 were -0.00199 and -0.00229, which indicated similar
trend for these two types of melanin. Therefore the purified melanin and
the standard melanin showed the same optical characteristic in UV-Vis
spectrophotometry.

(8) FT/IR Analysis

[0094] Melanin and KBr powder (Sigma) were baked at 60° C. oven for
1 h, mixed in the volume ratio of 1:19 and grounded into powder using an
agate mortar and pestle. The IR spectrum was scanned in the range of 4000
nm to 400 nm with a JASCO FTIR 4100 spectrophotometer (Jasco Corporation,
Tokyo, Japan) using pure KBr as blank. The spectra were analyzed with
KnowItAll° (BioRad, USA) to search the functional group, and
plotted with OriginPro 7.5 SR1 (USA) after the wavelength was converted
to μm.

[0095] The total complex structure of melanin is not completely known.
Only some monomer structures or model were provided (Kaxiras et al. 2006,
Moses et al. 2006). Melanin was reported to contain carbonyl, hydroxyl
and carboxyl groups. Referring to FIG. 15, the IR spectra of melanin from
transformant (MA05-169) and standard DOPA-melanin were quite similar
after FT/IR analysis. Both of them exhibited a peak near 3 um (˜3.2
um) which might be a --NH or --OH group. An additional peak at 6 um
(˜5.8 um) was also shown, which could be attributed to the
--C═O or double C bond (C═C) (Bonner and Duncan, 1962, Moses et
al. 2006). On the other hand, an extra peak at 3.3 um was exhibited only
in melanin from transformant but not in stand melanin, which might be
generated by a --CH2 or --CH3 group.

[0098] Referring to FIG. 17A, the virulence tests were performed using 3rd
instar larvae of P. xylostella (Diamondback moth), which was fed on 7-day
rape seedlings. The conidia of M. anisopliae wild type MA35505 and
transformant MA05-169 were washed out with 0.01% Tween 80 in the
concentration of 2×107 conidia/ml after cultivated in PDA
medium for 21 days. The 3rd instar larvae of P. xylostella were
soaked in the conidia-containing solution for 30 sec, and then dried on
filters. The larvae were reared on fresh rape seedlings at 27° C.
for 3 days. The mortality rates were determined by counting the living
larvae. The tests were performed in triplicate employing 50 larvae for 7
times independently. The virulence tests resulted in 80% mortality rates
for both groups (no significant difference) under no UV-radiation
condition.

(2) Effects of UV-Radiation on the Virulence of M. Anisopliae

[0099] Referring to FIGS. 17B and 17C, the conidia of M anisopliae wild
type MA35505 and transformant MA05-169 in the concentration of
2×107 conidia/ml were placed on Petri-dishes (5-cm in
diameter) respectively and irradiated with UV in the doses of 23.4 mJ/cm
and 46.8 mJ/cm2 without the lids, followed by the abovementioned
ways for infection, feeding and mortality rates determination. The tests
were performed in triplicate employing 50 larvae for 3 times
independently. The virulence test of wild type M. anisopliae resulted in
no death of P. xylostella, while transformant MA05-169 still showed a
mortality rate over 80% (FIG. 15). Therefore melanin-containing
transformant MA05-169 showed good virulence toward P. xylostella even
under UV-radiation condition.

(3) Germination Assay Against UV-Radiation Stress

[0100] Referring to FIGS. 18A to 18D, conidial germination assay of M.
anisopliae wild type MA35505 and transformant MA05-169 was undertaken in
UV-B radiation stress studies. Philips Ultraviolet-B TL 20W/12RS
(Holland) lamps without filter were used as the source of UV-B, which
emitted mostly UV-B, and some UV-C and UV-A. The distribution of spectra
and energy fluence were determined with a USB2000+ Miniature Fiber Optic
Spectrometer (Ocean Optics, USA) (FIG. 18A). 150-300 μl of the
conidial suspension in the concentration of 106-107 conidia/ml
was placed evenly on Petri-dishes and irradiated with 3 UV doses (0
mJ/cm, 23.4 mJ/cm and 46.8 mJ/cm2) in triplicate, and cultivated at
25° C. in the dark. Germination was observed for a period of 6,
10, 23, 28, 48 and 72 h post-inoculation under the 400×
magnification. Germinated (grew over the length of conidium) and
non-germinated conidia were counted in random. Random microscope fields
were selected until 100 conidia had been counted. Percent germination was
assessed in three random fields of view for each test.

[0101] As shown in FIGS. 18A to 18D, conidial germination rate of
transformant was obvious faster than that of wild type without UV-B
radiation (FIG. 18B). The germination rate of the transformant still
showed significant difference from that of wild type although the
germination rates of both strains were delayed by UV-B radiation. The
germination rate of wild type strain at 72 h post-inoculation was 10%
after irradiated at UV dose of 46.8 mJ/cm; while that of transformant was
58%, which was around 5 fold of the former. The germination rate of
transformant at 48 h post-inoculation was 2-fold to wild type strain at
UV dose of 23.4 mJ/cm (FIGS. 18C and 18D).

(3) Effects of Temperature on the Conidial Germination Rate of M.
Anisopliae

[0103] In brief, the proper temperature for conidial germination is around
20-30° C. for either wild type MA35505 or transformant MA05-169,
but transformant MA05-169 showed significantly higher germination rates
than wild type at the early periods of inoculation in any temperature.
The germination rate of wild type strain at 30° C. after
incubation for 6 h was 10%; while that of transformant was above 90%. And
the germination rate of transformant at 10° C. after incubation
for 15 h was around 50%; while wild type MA35505 did not germinate at
that time point. The germination rates between both strains were also
distinct at 15° C. after incubation for 15 h. The germination
rates between both strains were not significant after incubation for 24 h
at 20-30° C.; while the germination rates between both strains
became indifferent only till 48 h after inoculation at 35° C. And
the germination rates for transformant MA05-169 and wild type MA35505
were 97.1% and 69.5% respectively at 10° C. after 72 h.

(4) Effects of Water Activity on the Conidial Germination Rate of M.
Anisopliae

[0104] Glycerol was used to adjust the water activity (aw). Glycerol was
added in the amounts of 0, 4, 6, 10, 12, 20, 30 and 40 g to 100 ml of PDA
media. The water activities were determined with AquaLab® 3TE Water
Activity Meter (Washington, USA) to be 0.996, 0.991, 0.989, 0.986, 0.977,
0.967, 0.938 and 0.895 respectively. Water activities in 0.938 and 0.895
were not included in the table since the strains could not germinate.
Suspension of conidia were placed in PDA plates evenly, and cultivated at
25° C. incubator for 6, 13, 17, 24, 48 and 72 h to determine the
germination rates. Melanin-containing transformant MA05-169 showed
significantly higher germination rates than wild type at low water
activity media. The germination rate of transformant at water activity of
0.967 after incubation for 72 h was around 40%, while wild type MA35505
could not germinate at that condition (Table 4). Therefore the tolerance
toward drought was increased after transformation of melanin synthesizing
genes into M. anisopliae.

[0105] The results of the invention were subjected to statistical analysis
using Statistics Package for Social Science (SPSS). Standard curve was
plotted with Sigma Plot.

CONCLUSION

[0106] Based on the outcomes of the abovementioned experiments,
DHN-melanin synthesis could effectively increase the tolerance of
beneficial microorganisms against UV-radiation, extreme temperature or
low temperature, and drought condition. Protecting the microorganisms by
synthesis of melanin by themselves is different from the traditional way
of mixing the anti-UV compounds with microorganisms, which helps to
increase the survival of microorganisms and shows a faster and higher
infection ability to further increase the efficiency of beneficial
microorganisms. The present invention can also be applied in other
beneficial microorganisms and plants to effectively improve the tolerance
against environmental stress.